Development Of Indirect ELISA For Detection Of Antibodies Against Canine Rabies Virus

1. Purification of canine IgG and preparation of monoclonal antibodies against canine IgGCanine IgG was firstly purified by acid-ammonium sulfate method, and then further purified using chromatography with Superdex 200. SDS-PAGE analysis indicated that the canine IgG was pure and its concentration was 2.0 mg/ml. The 6-8 week-old female BALB/c mice were immunized three times with purified IgG and the spleen cells of the immunized mouse and SP2/0 cells were mixed in proportion of 10:1, and then were fused by 0.7 ml of 50% PEG 4000. Six hybridoma cell lines secreting monoclonal antibodies (McAbs) were produced by three-time limited dilution method. The titers of ascetic fluids of the 6 McAbs ranged from 104 to 106 as detected by indirect ELISA. The isotypes and subclasses of all 6 McAbs belonged to IgG1,κ. Western bolt analysis showed that the McAbs secreted by 3 hybridoma cell lines were against L chain of canine Immunoglobulin, and those secreted by the remaining 3 hybridoma cell lines were against H chain of canine Immunoglobulin.2. Preparation and characterization of monoclonal antibodies specific for nucleoprotein of rabies virusBALB/c mice were immunized with purified recombinant RV nucleoprotein (N). Splenocytes were fused with SP2/0 myeloma cells. Five hybridoma cell lines (1A4, 7H3, 7H6, 7F6 and 7F7) secreting monoclonal antibodies (McAbs) were produced by three-time limited dilution method. The titers of ascetic fluids of the 5 McAbs ranged from 104 to 106 as detected by indirect ELISA. The isotypes and subclasses of all 5 McAbs belonged to IgG1,κ. Western bolt analysis showed that these McAbs could specifically react with the recombinant RV N protein. However, only 7F6 and 7F7 McAbs recognized the N protein in the virus infected Vero cells in the indirect immunofluorescence assay.3. Preparation and characterization of monoclonal antibodies specific for glycoprotein of rabies virusFemale, 6-8 weeks old BALB/c mice were immunized with the purified recombinant RV glycoprotein. After 3 times of immunizations, the spleen cells of immunized mice were fused with SP2/0 cells. The antibodies were tested by an indirect ELISA assay. By 3 cycles of limited dilutions, we obtained 1 strain of hybridoma cells, which named 1D10. The titer of ascetic fluids was 2.56×104 as detected by indirect ELISA. The isotype and subclass of the McAb belonged to IgG1,κ. We identified the McAb using IFA with RV-infected Vero cells. The IFA titer of the McAb was 1:400.4. Purification and labeling process of anti-dog-IgG monoclonal antibodyThe cell line 3D4, which could secrete monoclonal antibody (McAb) against canine-IgG stably, was cultured, and then the ascites were produced in Balb/c mice. Two steps including acid-ammonium sulfate purified the McAb and Protein G. SDS-PAGE analysis indicated that the McAb was pure. The purified IgG was then labeled with peroxidase by the periodate method and further evaluated by Dot-ELISA assay. The combination rate of enzyme was 52.16% and the molar ratio of HRP and Ab was 1.6. When the HRP-labeled secondary antibody was diluted at 1:3000, it can still reacted with the 1:1024 diluted positive canine sera against rabies virus.5. An indirect ELISA based on the recombinant nucleoprotein for detection of canine antibodies against rabies virusThe N protein expressed in E.coli was used as the diagnostic antigen to develop a NP-based indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to canine rabies virus from canine sera. The optimal antigen concentration and serum sample dilution were set at 2μg/ml and 1:100; The optimal dilution of the conjugate was 1:2000; The 0.05 M Tris-HCl buffer at pH 8.5 was the best coating buffer; The 0.5% bovine serum albumin was the optimal blocking buffer; The phosphate buffer containing 0.1% bovine serum albumin was the best diluent; The optimal reaction time for serum samples or conjugate and chromatogenic substrate was 60 min and 10 min, respectively. The ELISA assay was confirmed to have good reproducibility, specificity and sensitivity by repetition and blocking test. Comparing with the SYNBIOTICS kit, the specificity, sensitivity and accuracy of the ELISA was 90.7%, 91.2% and 90.9%, respectively.